META: A computational tool for predicting metastable states in the folding pathways of proteins

The thermodynamic stabilities (∆G, free energy change) of proteins are being measured by traditional denaturation methods (∆GU, free energy of unfolding) and native hydrogen-deuterium (H/D) exchange method (∆GHX, free energy of exchange), in general. Estimation of an accurate ∆G for proteins at ambient conditions is indispensable to unambiguously address the folding events of the proteins, which may pave the way of designing de novo therapeutic peptides with defined functions. However, the ΔGU and the ΔGHX of proteins are not in good agreement, in general. Herein, we have developed a META program, which accounts the discrepancies between the ΔGU and the ΔGHX of proteins by predicting the possible existence of higher energy metastable states in the folding kinetics of proteins under native conditions by systematically analysing residue-specific free energies of the proteins. The robustness of the program on analyzing the conformational stabilities of proteins has been validated using experimental data available in the literature and the implication of the program on medicinal chemistry is also discussed. The program is publicly available at http://feat.sastra.edu/meta.html. Introduction: Each protein adopts a specific welldefined three-dimensional (3D) structure with unique stability, which is important for its biological activities. The relationships between the conformations of such proteins and their stabilities have intrigued researchers for many decades [1]. The ∆GU of proteins is calculated by fitting the data of the unfolded population (U) of proteins with respect to denaturant concentration or temperature, to an appropriate two-state model equation [2, 3]. The ∆GHX of proteins is determined using hydrogen-deuterium (H/D) exchange method in conjunction with NMR technique [4]. Accurate estimation of ∆GU and ∆GHX for proteins at ambient conditions is indispensable to unambiguously address the thermodynamic and kinetic events of the proteins, respectively [5 7]. Under similar experimental conditions, it is expected that ∆GU and ∆GHX of protein should be same. But many proteins do not follow this tradition, for which the probable reason could be presence of metastable states in the folding pathways of proteins [8, 9]. Though the discrepancies in a few proteins have been experimentally addressed [10, 11], the problem left unaddressed in most cases. Moreover, unique computational programs by which such discrepancies can be systematically addressed have not been developed to date. In the present study, we describe a program (META), which addresses the discrepancies between the ∆GHX and ∆GU of proteins on predicting the possible existences of higher energy metastable states in the folding pathways of proteins under native conditions. The uniqueness of the META to address the discrepancies between the ∆GHX and ∆GU of proteins is validated using experimental data available in the literature and its applications on exploring energy landscape of proteins and on designing therapeutic peptides are also brought into fore. Methods: 2.1 META algorithm META algorithm has been implemented using PERL scripting language [12]. The program requires only three inputs from the user: PDB file of proteins, residue specific ∆GHX and free energy of unfolding, ∆GU of the proteins. In outline, the program has five stages for each complete cycle. In the first stage, the ∆GHX of proteins is calculated by averaging out to four largest residue-specific ∆GHX of the proteins [13]. In the second stage, Tambi Richa et al /J. Pharm. Sci. & Res. Vol.3(9), 2011,1486-1490